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NEUROPHARMACOLOGY

Effects of Prenatal Ethanol Exposure on the Excitability of Ventral Tegmental Area Dopamine Neurons in Vitro

Research Institute on Addictions, State University of New York at Buffalo, Buffalo, New York

Received for publication June 6, 2006
Accepted August 9, 2006.

	Abstract

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Prenatal ethanol exposure leads to a persistent reduction in the number of spontaneously active dopaminergic (DA) neurons (DA neuron population activity) in the ventral tegmental area (VTA) in developing and adult animals. This effect might contribute to the dysfunction of the mesolimbic/cortical DA system and attention problems in children with fetal alcohol spectrum disorders. To characterize the underlying cellular mechanism for prenatal ethanol exposure-induced reduction in VTA DA neuron population activity, we used the whole-cell patch-clamp technique to study the membrane properties of putative VTA DA neurons in brain slices in 2- to 3-week-old control and prenatal ethanol-exposed animals. The results show that prenatal ethanol exposure did not impair the spontaneous pacemaker activity in putative VTA DA neurons but reduced the frequency of evoked action potentials. In addition, prenatal ethanol exposure led to a reduction in hyperpolarization-induced cation current (I_h) and an up-regulation of somatodendritic DA autoreceptors. The above prenatal ethanol exposure-induced changes could decrease the excitability of VTA DA neurons. However, they do not seem to play a role in reduced VTA DA neuron population activity in vivo, an effect thought to be mediated by excessive excitation leading to depolarization inactivation. Taken together, the above results indicate that prenatal ethanol exposure-induced reduction in VTA DA neuron population activity in vivo is not caused by changes in the intrinsic pacemaker activity or other membrane properties and could instead be caused by altered inputs to VTA DA neurons.

The prenatal ethanol exposure-induced dysfunctions in the mesolimbic/cortical DA system include reduced DA synthesis, uptake sites, receptor binding sites, and DA metabolites in both DA neuron cell body and terminal areas (Rathbun and Druse, 1985; Cooper and Rudeen, 1988; Druse et al., 1990; Szot et al., 1999). Dopaminergic neurons also have smaller cell bodies and retarded dendritic growth in prenatal ethanol-exposed animals (Shetty et al., 1993). In addition, the sensitivity of both pre- and postsynaptic DA receptors and DA receptor-mediated behaviors are affected by prenatal ethanol exposure (Shen et al., 1995; Hannigan, 1996). In the past few years, we have studied the impact of prenatal ethanol exposure on the electrical activity of midbrain DA neurons using the in vivo single-unit extracellular recording technique. The results of these studies show that prenatal ethanol exposure leads to a persistent reduction in the electrical activity of midbrain DA neurons located in the substantia nigra and VTA (Shen et al., 1999; Xu and Shen, 2001; Choong and Shen, 2004a,b; Shen and Choong, 2006). The electrical activity of DA neurons is critical in controlling the synthesis and release of DA (Gonon and Buda, 1985; Suaud-Chagny et al., 1992). Therefore, we suggest that prenatal ethanol exposure-induced reduction in the electrical activity of DA neurons could contribute to decreased DA function characterized in previous biochemical studies (Rathbun and Druse, 1985; Cooper and Rudeen, 1988; Druse et al., 1990; Szot et al., 1999).

The prenatal ethanol exposure-induced reduction in the electrical activity in DA neurons is mainly reflected in a prominent decrease in the number of spontaneously active DA neurons (DA neuron population activity). A small, but significant, decrease in the firing rate is often but not always observed (Shen et al., 1999; Choong and Shen, 2004a,b; Shen and Choong, 2006). Furthermore, the reduced DA neuron population activity is not caused by a permanent loss of DA neurons (Shen et al., 1999) and can be reversed by acute administration of DA receptor agonists (Shen et al., 1999; Xu and Shen, 2001; Choong and Shen, 2004b; Shen and Choong, 2006). Dopaminergic receptor agonists in normal animals exert inhibitory effect on DA neurons and reduce their population activity. Therefore, a reversal in reduced population activity (increase) by DA receptor agonists suggests that the reduced population activity in prenatal ethanol-exposed animals is caused by an increase in the number of quiescent DA neurons in the state of depolarization inactivation due to excessive excitation (Grace et al., 1997).

Despite the possibility that prenatal ethanol exposure-induced reduction in VTA DA neuron population activity could contribute to the dysfunction of midbrain DA systems and the etiology of attention problems in individuals with FASDs, the underlying cellular mechanism(s) has not been characterized. The electrical activity of DA neurons is controlled by membrane properties responsible for the excitability and the intrinsic pacemaker activity (Kang and Kitai, 1993a,b; Grace et al., 1997), as well as input modulation (Kalivas, 1993; Kitai et al., 1999). Prenatal ethanol exposure could affect either or both factors to reduce VTA DA neuron population activity in vivo. In the present study, we used the in vitro patch-clamp technique to investigate whether the intrinsic membrane properties of DA neurons in the VTA in developing rats were altered by prenatal ethanol exposure. We focused on VTA DA neurons because these neurons are the origin of the mesolimbic/cortical DA system, which plays an important role in attention (Denney, 2001; Davids et al., 2003).

	Materials and Methods

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Prenatal Ethanol Treatment and Cross-Fostering. The procedures of prenatal ethanol treatment and cross-fostering are described in detail in previous studies (Choong and Shen, 2004a,b; Shen and Choong, 2006). In brief, timed-pregnant Sprague-Dawley rats were purchased from Harlan Sprague-Dawley (Indianapolis, IN) and were delivered to the animal facility on gestational day 6. Ethanol was administered via intragastric intubation to dams from gestation day 8 through 20 with a daily dose of 0 or 6 g/kg ethanol (20% w/v in 0.9% saline) during weekdays by two intubations at 0 or 3 g/kg. A single daily dose of 0 or 4 g/kg ethanol was given during weekends. The control dams received the same volume of a sucrose solution (30% w/v in 0.9% saline) to substitute for ethanol isocalorically. Dams in the control group were pair-fed with ethanol-treated dams. Dams also received thiamine injections twice a week (8 mg/kg intramuscularly). On postnatal day 1, pups were randomly grouped to litters of 10 while maximizing the number of males in each litter. The litters were then transferred to surrogate dams that did not receive any treatment and had delivered 2 days earlier. Litters were weaned and weighed on postnatal day 21. To control for possible litter effect, no more than three male littermates were used in the present study.

Preparation of Midbrain Slices. Midbrain slices were prepared as described previously (Johnson and North, 1992). In brief, animals were anesthetized with halothane and killed by decapitation. The brain was removed and cooled in ice-cold oxygenated Ringer's solution containing 124 mM NaCl,2.3 mM KCl, 1.3 mM MgSO₄, 2.5 mM CaCl₂, 1.0 mM NaH₂PO₄, 26.2 mM NaHCO₃, and 11 mM glucose. A block containing the midbrain was isolated and fixed to a stage with cyanoacrylate glue. Two to three coronal midbrain slices (400 µm) containing the VTA were obtained using a Vibratome (Lancer series 1000; Ted Pella, Irvine, CA). The slices were transferred to an incubation chamber and incubated in oxygenated Ringer's solution at room temperature for at least 1 h. The slices were transferred one at a time to the recording chamber and continuously perfused with normal Ringer's solution (2 ml/min) saturated with 95% O₂-5% CO₂ at 30 ± 1°C.

The above prenatal ethanol treatment and the surgical procedures were conducted in accordance with the National Institutes of Health and American Association for Accreditation of Laboratory Animal Care guidelines. Procedures were approved by the Institutional Animal Care and Use Committee at the University of Buffalo.

Whole-Cell Patch-Clamp Recording. Whole-cell recordings of VTA DA neurons were performed using the tight-seal patch-clamp technique. Recording electrodes (resistance 4–7 M) were pulled from a 1.2-mm (outside diameter) borosilicate glass pipette (World Precision Instruments, Inc., Sarasota, FL) and filled with an internal solution of the following composition: 137 mM potassium gluconate, 5 mM KCl, 5 mM NaCl, 1 mM MgCl₂, 10 mM HEPES, 0.02 mM EGTA, 2 mM Na₂-ATP, and 0.5 mM Na-GTP. The pH was adjusted to 7.3 to 7.4 with KOH. Electrical signals were amplified with an Axoclamp 2B amplifier (Axon Instruments, Foster City, CA). Voltage and current were filtered at 5 to 10 kHz and recorded on-line using an Intel processor-based PC equipped with a 12-bit analog-to-digital converter under the control of pClamp 7.0 software (Axon Instruments) and/or a paper chart recorder (model TA240; Gould Instruments, Valley View, OH). The neurons were recorded in the medial and posterior VTA, which could be visualized as areas medial to the medial terminal nucleus of the accessory optic track or areas medial to medial lemniscus. Putative VTA DA neurons were identified by a prominent voltage sag caused by the hyperpolarization activated cation current (I_h) in response to positive current injections in the current-clamp mode. Some DA neurons were further identified by dopamine-induced membrane hyperpolarization. Although the majority of the tyrosine hydroxylase positive neurons can be identified with the presence of I_h or DA-induced hyperpolarization, neither of the criteria can reach 100% accuracy (Cameron et al., 1997; Margolis et al., 2003). Therefore, neurons identified by the presence of I_h only represent putative VTA DA neurons.

Spontaneous action potential widths and amplitudes for individual VTA DA neurons were measured at the firing threshold. Input resistance was estimated in the current-clamp mode by the voltage drop when a negative d.c. current was injected (–0.2 nA; 500 ms) at –60 mV. The frequency of evoked action potentials and the latency to spike onset were also studied by positive d.c. current injections (from 0.05 to 0.25 nA; increment of 0.05 nA). We also measured the amplitudes of negative d.c. currents required to hyperpolarize the membrane potential to –60 mV in a subset of VTA DA neurons. This index was to serve as an indirect estimate for possible differences in the resting membrane potential of VTA DA neurons, which could not be directly measured in VTA DA neurons displaying spontaneous action potentials.

To examine the effect of prenatal ethanol exposure on I_h in VTA DA neurons, the I-V activation curves were obtained in the voltage-clamp mode with 0.5-s hyperpolarizing steps (–50 to –140 mV; increment of 10 mV), whereas the membrane potential was held at –40 mV. Series resistance was compensated to 80% in the voltage-clamp experiments using the bridge circuit of the amplifier, and the settling time of the membrane current in response to 10 mV hyperpolarization was optimized to <5 ms. The amplitude of I_h at each hyperpolarizing step was obtained by subtracting the instantaneous current amplitude from the steady-state current amplitude. The normalized I_h curve for each VTA DA neuron was obtained by fitting the I_h amplitude with the Boltzmann equation: Y = Y_max/[1 – exp(V – V₅₀)/s], where Y_max is the maximal I_h current amplitude (set at 1), V is the membrane potential, V₅₀ is the half-activation potential, and s is the slope. The normalized I_h amplitudes Y at different membrane potentials (V) and the half-activation voltage (V₅₀) in each VTA DA neuron were then determined.

To examine the function of the somatodendritic DA receptors, VTA DA neurons were hyperpolarized and maintained at –60 mV by negative d.c. injections in the current-clamp mode. Sodium metabisulfite (100 µM; Sigma Aldrich, St. Louis, MO), which had no effect on the excitability of VTA neurons, was added to bath to prevent dopamine oxidization. Dopamine (dopamine hydrochloride; Sigma Aldrich) was bath-applied at 1, 3, 10, 30, 100, and 200 µM, and the membrane hyperpolarization by dopamine was recorded in the presence of tetrodotoxin (1 µM; Alomone Laboratories, Jerusalem, Israel) with a paper chart recorder. Each neuron received two to four different doses of dopamine. Data were used only when membrane potential returned to the predopamine level during the washout period. Results from previous studies have shown that hyperpolarization induced by bath-applied dopamine in DA neurons in brain slices is mediated by the activation of somatodendritic DA autoreceptors which are the D₂-like receptors (Momiyama et al., 1993a,b).

Data Analysis. Data were analyzed with Statistica (StatSoft, Groningen, Netherlands) and plotted with Origin (Microcal Software, Northampton, MA). Numerical data are presented as means ± S.E.M. Independent t test, ANOVA/MANOVA with or without repeated measures were used for statistical comparisons. MANOVA was used for comparisons of action potential characteristics that were intercorrelated. Fisher's least significant difference post hoc comparison was used for post hoc comparisons. P < 0.05 was considered to be statistically significant.

	Results

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Identification of VTA DA Neurons. Putative VTA DA neurons were identified on the basis of the presence of I_h in the current-clamp mode (Fig. 1A) (Johnson and North, 1992; Cameron et al., 1997) as a prominent voltage "sag" when a negative d.c. current step was administered at –60 mV (Fig. 1A). In accordance with the electrophysiological characteristics of VTA DA neurons, the great majority of putative VTA DA neurons also displayed slow pacemaker action potentials (<2 Hz) (Fig. 1B). Dopamine was bath-applied to 27 putative VTA DA neurons; membrane hyperpolarization was observed in all of these neurons when dopamine concentration was 10 µM (Fig. 1C).

View larger version (9K): [in this window] [in a new window]   Fig. 1. VTA DA neurons identified in vitro. A and B, putative VTA DA neurons in vitro were identified by the presence of Ih—a prominent voltage "sag" in response to a negative current injection (–0.2 nA, 500 ms, membrane potential held at –60 mV). Neurons identified by Ih also showed spontaneous pacemaker activity and slow after hyperpolarization after each action potential typical of DA neurons. C, when DA was bath applied (100 µM), neurons identified by Ih were hyperpolarized.

The characteristics of spontaneous action potentials including firing rate, action potential threshold, peak-to-peak amplitude, and action potential peak potential from individual neurons were compared between control and prenatal ethanol-exposed animals. In addition, age of the animals at the time of the recording (i.e., 2 or 3 weeks old) was also considered. The results showed that neither prenatal ethanol exposure nor age influenced the characteristics of spontaneous action potentials (two-way MANOVA) (Table 1).

Effects of Prenatal Ethanol Exposure on Evoked Action Potentials. To verify whether prenatal ethanol exposure altered the evoked activities in putative VTA DA neurons, the frequency of evoked action potentials was studied in the current-clamp mode by injecting positive currents (0.05– 0.25 nA) at –60 mV. The input-output curves were compared between control (n = 35) and prenatal ethanol-exposed animals (n = 38) at different ages (Fig. 2A). Lower frequency responses in putative VTA DA neurons were recorded from prenatal ethanol-exposed animals. This effect was reflected in a significant interaction effect between group and evoked current amplitude (three-way ANOVA with repeated measures, F_4,276 = 3.07, P < 0.05) (Fig. 2B). On the other hand, age was not a determining factor of evoked action potential frequency of putative VTA DA neurons (three-way ANOVA with repeated measures, Fig. 2B).

View larger version (17K): [in this window] [in a new window]   Fig. 2. The effect of prenatal ethanol exposure on evoked action potentials. A, representative recording traces of evoked action potentials in putative VTA DA neurons in response to positive current injections in control (left) and prenatal ethanol-exposed (right) animals. B, input-output (I-V) curves showing that prenatal ethanol exposure significantly reduced the frequency of evoked action potentials. C, plot of latency to spike onset showing that prenatal ethanol exposure led to significantly greater latency to spike onset.

Effects of Prenatal Ethanol Exposure on I_h Current. Voltage-clamp recordings were performed on putative VTA DA neurons in control (n = 25) and prenatal ethanol-exposed animals (n = 17) to examine the effects of prenatal ethanol exposure on I_h. The magnitude of the I_h current increased as the membrane was hyperpolarized to more negative potentials (two-way ANOVA with repeated measures, F_9,378 = 269.96, P < 0.001) (Fig. 3A, B, and C). Analysis of current versus voltage (I-V) plots (Fig. 3B) revealed that prenatal ethanol exposure significantly reduced the magnitude of I_h current (two-way ANOVA with repeated measures, F_9,378 = 6.20, P < 0.001). Note that the differences were more apparent in membrane potentials more negative than –90 mV (Fisher's least significant difference post hoc comparison, P < 0.05). On the other hand, the analysis of normalized I-V curves of I_h showed no differences between the control and prenatal ethanol-exposed animals (two-way ANOVA with repeated measures) (Fig. 3C). The V₅₀ values for putative VTA DA neurons recorded from control and prenatal ethanol-exposed animals were not different (t test; control: –103.4 ± 1.3 mV; prenatal ethanol exposure: –104.2 ± 1.4 mV).

View larger version (33K): [in this window] [in a new window]   Fig. 3. Effects of prenatal ethanol exposure on Ih. A, representative voltage-clamp recording traces of Ih in putative VTA DA neurons from a control (left) and a prenatal-ethanol exposed (right) animal. Membrane potential was held at –40 mV and stepped down to the range of –50 to –140 mV. B, current versus voltage plot depicting Ih amplitude at membrane potential more negative than –90 mV was significantly smaller in prenatal ethanol-exposed animals. C, no differences in normalized current versus voltage plots of Ih in putative VTA DA neurons recorded from control and prenatal ethanol-exposed animals. *, P < 0.05, significant difference in Ih between control and prenatal ethanol-exposed animals.

View larger version (31K): [in this window] [in a new window]   Fig. 4. The effect of prenatal ethanol exposure on somatodendritic DA autoreceptor function. A, representative recording traces in the current-clamp mode for the hyperpolarizing effect of bath-applied dopamine on putative VTA DA neurons. B, prenatal ethanol exposure significantly reduced the maximal inhibition by dopamine and shifted the dose-response curve to the left. C, the normalized dose-response curves reveal a significant left shift in prenatal ethanol-exposed animals, which indicates an up-regulation in somatodendritic DA autoreceptors in putative VTA DA neurons. *, P < 0.05, significant difference in dopamine-induced hyperpolarization between control and prenatal ethanol-exposed animals.

	Discussion

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The major finding of the present study is that prenatal ethanol exposure did not alter the intrinsic pacemaker activity of putative VTA DA neurons observed in brain slices in developing animals (2–3 weeks old). The great majority (97%) of putative VTA DA neurons recorded from the control and prenatal ethanol-exposed animals displayed a spontaneous pacemaker pattern of action potentials. This result is consistent with the observations from previous studies in DA neurons recorded in brain slices (Lacey et al., 1989; Grace, 1991; Johnson and North, 1992; Kang and Kitai, 1993a,b; Cameron et al., 1997). The spontaneous firing rates and action potential characteristics of putative VTA DA neurons were similar in control and prenatal ethanol-exposed animals. In addition, there was no age-dependent effect on the proportion of putative VTA DA neurons expressing spontaneous action potentials or their firing rate in either control or prenatal ethanolexposed animals. The above results contrast with those from our previous in vivo studies in the following manner (Shen et al., 1999; Xu and Shen, 2001; Choong and Shen, 2004a; Shen and Choong, 2006). First, in control animals, VTA DA neurons in vivo display burst firing activity and age-dependent changes in the population activity and firing rate (Choong and Shen, 2004a). Second, these normal age-dependent changes are altered in prenatal ethanol-exposed animals. The age-dependent decrease in VTA DA neuron population activity is enhanced, whereas the age-dependent increase in firing rate is reduced. This results in a persistent decrease in the population activity and a slight reduction in the firing rate of VTA DA neurons in prenatal ethanol-exposed animals. In the present in vitro study, neither a normal age-dependent effect nor a prenatal ethanol exposure-induced effect on the spontaneous action potentials of putative VTA DA neurons is observed. Therefore, we suggest that the membrane properties responsible for the intrinsic pacemaker activity do not play a major role in the age-dependent development or prenatal ethanol exposure-induced changes in the population activity or firing rate of VTA DA neurons observed in vivo.

In contrast to spontaneous action potentials, we observed that the frequency of evoked action potentials of putative VTA DA neurons was significantly reduced in prenatal ethanol-exposed animals. In addition, this effect was not mediated by a decrease in input resistance, which was actually higher in putative VTA DA neurons recorded from prenatal ethanol-exposed animals—an effect possibly due to smaller sizes of cell bodies (Shetty et al., 1993). The reduced frequency in evoked action potentials in prenatal ethanol-exposed animals may lead to a decreased firing rate in response to excitatory inputs (Kalivas, 1993; Kitai et al., 1999) and contribute to the small, but significant, reduction in VTA DA neuron firing rate observed in vivo (Choong and Shen, 2004a; Shen and Choong, 2006). At the present time, the cause of decreased evoked action potential frequency is not clear. A recent study showed that increased transient potassium current (I_A) in cultured midbrain DA neurons can lead to reduced evoked action potential frequency (Hahn et al., 2006). However, this study also showed that increased I_A reduces the spontaneous pacemaker activity, a result not observed in the current study. Therefore, further studies are required to delineate whether I_A or another ionic current is responsible for reduced evoked action potential frequency in prenatal ethanol-exposed animals.

In the present study, we also studied I_h because it plays an important role in generating spontaneous pacemaker activity in neurons (Pape, 1996). A reduced firing rate has been observed in DA neurons in the presence of a specific I_h blocker (Seutin et al., 2001), suggesting that I_h is actively involved in the generation of the spontaneous pacemaker activity. In addition, I_h is a target of ethanol. Acute ethanol facilitates I_h (Brodie and Appel, 1998; Okamoto et al., 2006). The result from the present study shows that the maximal current of I_h, but not the V₅₀, was reduced in putative VTA DA neurons recorded from prenatal ethanol-exposed animals. This finding is similar to the results obtained from developing animals undergoing withdrawal from repeated ethanol treatment (Okamoto et al., 2006) and indicates that the reduction in I_h could be a persistent and common consequence after repeated ethanol exposure during development. At the present time, the cause for the prenatal ethanol exposure-induced decrease in I_h is not known. Based on previous studies in cortical pyramidal neurons (Magee, 1998), I_h channels are largely located in distal dendrites. One possible mechanism mediating decreased maximal current of I_h could be a decreased number of I_h channels resulting from retarded dendritic branching in DA neurons in prenatal ethanol-exposed animals (Shetty et al., 1993). Our results show that I_h amplitudes between –60 and –90 mV did not differ between the control and prenatal ethanol-exposed animals. In addition, prenatal ethanol exposure did not affect the pacemaker activity of putative VTA DA neurons. Therefore, it is unlikely that reduced maximal I_h in prenatal ethanol-exposed animals plays a major role in reduced VTA DA neuron population activity in vivo.

We also observed an up-regulation of somatodendritic DA autoreceptors in putative VTA DA neurons recorded from prenatal ethanol-exposed animals. This effect was reflected in reduced EC₅₀ and increased V_max and suggests that both the sensitivity and number of somatodendritic autoreceptors are increased after prenatal ethanol exposure. The up-regulation in somatodendritic DA autoreceptors observed in the present study is consistent with that obtained in a previous in vivo study in adult animals (Shen et al., 1995). Apparently, the up-regulation of the somatodendritic DA autoreceptors in prenatal ethanol-exposed animals takes place during early postnatal development and persists into adulthood. The up-regulation of somatodendritic DA receptors may have complex effects on the excitability of VTA DA neurons. First, acute activation of these receptors can decrease the activity of DA neurons via the opening the potassium channels mediated by I_A and provide an immediate negative feedback control of DA neuron activity (Chiodo, 1988; Liu et al., 1994; results from the present study). Results from a recent study in cultured DA neurons suggest that tonic stimulation of somatodendritic DA autoreceptors and the activation of I_A play an important role in the maintenance of the pacemaker activity in DA neurons (Hahn et al., 2006). Chronic blockade of these receptors leads to an increase in the current density of I_A and a reduction in the pacemaker activity in DA neurons. Therefore, it is conceivable that the up-regulation of the somatodendritic DA receptors in prenatal ethanol-exposed animals could also modify I_A and contributes to reduced VTA DA neuron population activity in vivo. However, the results from the present study show a lack of change in the spontaneous pacemaker activity of putative VTA DA neurons in prenatal ethanol-exposed animals. Furthermore, stimulating the somatodendritic DA receptors in prenatal ethanol-exposed animals actually reverses the reduction (increase) in the population activity of DA neurons (Shen et al., 1999; Xu and Shen, 2001; Choong and Shen, 2004b; Shen and Choong, 2006). Therefore, the up-regulation of somatodendritic DA receptors does not seem to play a critical role in reduced VTA DA neuron population activity in vivo.

The results from the present study show that prenatal ethanol exposure does not alter the spontaneous pacemaker activity of putative VTA DA neurons. On the other hand, prenatal ethanol exposure leads to other changes in membrane properties of these neurons including reduced evoked action potential frequency, decreased I_h, and the up-regulation of the somatodendritic autoreceptors. These changes could potentially lead to decreased excitability of VTA DA neurons and underlie the reduced population activity of VTA DA neurons in vivo. However, our previous findings suggest that increased excitation to the extent of depolarization inactivation, instead of increased inhibition, leads to reduced VTA DA neuron population activity (Shen et al., 1999; Xu and Shen, 2001; Choong and Shen, 2004b; Shen and Choong, 2006). Therefore, it is unlikely that the above changes in membrane properties contribute to reduced population activity in VTA DA neurons in vivo. Some of the above effects might actually represent compensatory responses to counter the excessive excitation in VTA DA neurons. Based on the results from the present study, we suggest that prenatal ethanol exposure-induced reduction in VTA DA neuron in vivo is caused by altered input modulation. This notion is consistent with findings from previous in vivo studies showing that intact inputs are required for the manifestation of reduced population activity in VTA DA neurons due to depolarization inactivation after chronic antipsychotic treatment (Grace et al., 1997). In vitro studies currently performed in our laboratory aiming at the synaptic transmission onto VTA DA neurons might be able to elucidate the cellular mechanism for the prenatal ethanol exposure-induced reduction in VTA DA population activity in vivo.

	Acknowledgements

 
We thank Patricia Leach for excellent technical support for the prenatal ethanol treatment.

	Footnotes

 
This research was supported by Grant AA 12435 from the National Institute on Alcohol Abuse and Alcoholism.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.109041.

ABBREVIATIONS: FASD, fetal alcohol spectrum disorder; DA, dopamine; VTA, ventral tegmental area; ANOVA, analysis of variance; MANOVA, multiple analysis of variance; d.c., direct current.

Address correspondence to: Dr. Roh-Yu Shen, Research Institute on Addictions, 1021 Main St., Buffalo, NY 14203. E-mail: shen{at}ria.buffalo.edu

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